METHODS AND DEVICES FOR DETECTION OF PERIPHERAL MEMBRANE PROTEIN INTERACTIONS USING NONLINEAR OPTICAL TECHNIQUES
Methods and devices for identifying agents that block binding or activation of peripheral membrane proteins are disclosed.
1 .- 46 . (canceled)
47 . A method for detecting binding of a candidate peripheral membrane protein to a supported membrane, the method comprising:
a) providing a substrate having a first substrate surface comprising a supported membrane, wherein the supported membrane comprises one or more lipid or protein components;
b) contacting the supported membrane with the candidate peripheral membrane protein, wherein the candidate peripheral membrane protein is labeled with a nonlinear-active label;
c) illuminating the first substrate surface with excitation light of at least one fundamental frequency, wherein the excitation light is provided by at least one light source; and
d) detecting light generated by the nonlinear-active label as a result of the illumination in step c) both before and after contacting the supported membrane with the candidate peripheral membrane protein, wherein a change in a physical property of the light detected before and after contacting the supported membrane with the candidate peripheral membrane protein indicates that the candidate peripheral membrane protein binds to the supported membrane or a component thereof;
wherein the candidate peripheral membrane protein is a protein comprising a structural domain selected from the group consisting of annexin, synapsin I, synuclein, gamma-carboxyglutamate (GLA)-domains of blood coagulation components, cytoskeletal proteins spectrin, and α-actinin-2.
48 . The method of claim 47 , wherein the supported membrane comprises a supported lipid bilayer.
49 . The method of claim 47 , further comprising adjusting the relative percentages of two or more lipid components in the supported lipid bilayer to enhance the degree of binding of the peripheral membrane protein to the supported lipid bilayer.
50 . The method of claim 47 , wherein the supported membrane comprises an artificial membrane.
51 . The method of claim 50 , wherein the artificial membrane has been modified by attachment of one or more lipid, protein, or membrane protein components.
52 . The method of claim 47 , wherein the light generated by the nonlinear-active label is coherent.
53 . The method of claim 47 , wherein the nonlinear-active label comprises a second harmonic-active, sum frequency-active, or difference frequency-active label.
54 . The method of claim 47 , wherein the one or more nonlinear-active labels are nonlinear-active unnatural amino acids that have been incorporated into the peripheral membrane protein.
55 . The method of claim 47 , wherein the first substrate surface comprises an array of supported lipid bilayers or artificial membranes localized in discrete regions of the first substrate surface.
56 . The method of claim 55 , wherein one or more lipid components of the supported lipid bilayer comprise lipids selected from the group consisting of diacylglycerol, phosphatidic acid (PA), phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylinositol phosphate (PIP), phosphatidylinositol biphosphate (PIP2), phosphatidylinositol triphosphate (PIP3), ceramide phosphorylcholine (sphingomyelin; SPH), ceramide phosphorylethanolamine (sphingomyelin; Cer-PE), ceramide phosphoryllipid, cholesterol, or any combination thereof.
57 . The method of claim 48 , wherein one or more protein components of the supported lipid bilayer comprise proteins or subdomains thereof selected from the group consisting of G-protein coupled receptors (GPCRs), transmembrane receptors, receptor tyrosine kinases, ion channel proteins, cytochrome P450 enzymes (CYPs), transport proteins, photosynthetic reaction centers, lipid-anchored proteins, or any combination thereof.
58 . The method of claim 47 , wherein the candidate peripheral membrane protein or a subdomain thereof is selected from the group consisting of extracellular matrix proteins, protein kinases, transmembrane receptor regulatory proteins, ion channel regulatory proteins, cytochrome proteins, lipid-anchored proteins, or any combination thereof.
59 . The method of claim 47 , wherein the peripheral membrane protein is contacted with a test agent prior to or simultaneously with contacting the supported lipid bilayer or artificial membrane with the peripheral membrane protein.
60 . The method of claim 47 , wherein the peripheral membrane protein is contacted with a test agent after contacting the supported lipid bilayer or artificial membrane with the peripheral membrane protein.
61 . The method of claim 47 , further comprising comparing the change in a physical property of the detected light observed upon contacting the peripheral membrane protein with a test agent to that observed upon contacting the peripheral membrane protein with a control.
62 . The method of claim 47 , wherein one or more lipid components or one or more protein components of the supported lipid bilayer or artificial membrane are also labeled with a nonlinear-active label.
63 . The method of claim 47 , wherein one or more lipid components or one or more protein components are labeled with a nonlinear-active label that is the same as that used to label the peripheral membrane protein.
64 . The method of claim 47 , wherein one or more lipid components or one or more protein components are labeled with a nonlinear-active label that is different from that used to label the peripheral membrane protein.
65 . The method of claim 47 , wherein the candidate peripheral membrane protein is a protein comprising a subdomain selected from the group consisting of C1 domains, C2 domains, PX domains, Fab1 domains, YOTB domains, Vac1 domains, EEA1 domains, FYVE domains, ENTH domains, ANTH domains, BAR domains, FERM domains, PDZ domains, Tubby domains, or any combination thereof.